Polyal drug conjugates comprising variable rate-releasing linkers

ABSTRACT

Polyal-Drug conjugates comprising a variable rate-releasing linker are described along with methods of making such conjugates. Uses for such Polyal-Drug conjugates is also described.

PRIORITY

This application claims the benefit of U.S. Provisional Application No.61/181,926, filed May 28, 2009. The entire disclosure of thatapplication is relied on and incorporated into this application byreference.

INCORPORATION BY REFERENCE

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights whatsoever.

FIELD

This application is directed to polymer-drug conjugates. In particular,this application is directed to polyal-drug conjugates comprisingvariable rate-releasing linkers, methods for using the same, and methodsfor designing the same.

BACKGROUND

Traditionally, pharmaceuticals have primarily consisted of smallmolecules that are dispensed orally (as solid pills and liquids) or asinjectables. Over the past three decades, however, sustained releaseformulations (i.e., compositions that control the rate of drug deliveryand allow delivery of the therapeutic agent at the site where it isneeded) have become increasingly common and complex. Nevertheless, manyquestions and challenges regarding the development of new treatments, aswell as the mechanisms with which to administer them, remain to beaddressed.

Although considerable research efforts in this area have led tosignificant advances, drug delivery methods/systems that have beendeveloped over the years and are currently used, still exhibit specificproblems that require some investigating. For example, many drugsexhibit limited or otherwise reduced potencies and therapeutic effectsbecause they are generally subject to partial degradation before theyreach a desired target in the body. Once administered, sustained releasemedications deliver treatment continuously, e.g. for days or weeks,rather than for a short period of time (hours or minutes). One objectivein the field of drug delivery systems, is to deliver medications intactto specifically targeted areas of the body through a system that cancontrol the rate and time of administration of the therapeutic agent bymeans of either a physiological or chemical trigger. The rate of releaseof a drug from a polymeric conjugate can play a very significant role inaltering the properties of the released drug, including having effectson the overall efficacy of the released drug, the duration of action ofthe released drug, the frequency of dosing required, the toxicity of thereleased drug, the biodistribution of the released drug, and the overallpharmacokinetic and pharmacodynamic properties of the released drug. Forexample, a slow, continuous release of a drug from a polymeric conjugatecan mimic the effect of a slow, continuous infusion of the drug. Such adelivery can be beneficial, for example, with a drug-release productwhich has an inherently short-half life, and therefore would requiremuch more frequent dosing if administered directly. Furthermore, apolymer conjugate of a drug release product could be designed to alterthe C_(max) of a drug-release product; by carefully designing a polymerconjugate with an appropriate release half-life, one can target aC_(max) value such that it falls within a desired therapeutic window,for example, lower than a value known to have an associated toxicity,while maintaining a therapeutic level of the drug-release product.

Over the past decade, materials such as polymeric microspheres, polymermicelles, soluble polymers and hydrogel-type materials have been shownto be effective in enhancing drug targeting specificity, loweringsystemic drug toxicity, improving treatment absorption rates, andproviding protection for pharmaceuticals against biochemicaldegradation, and thus have shown great potential for use in biomedicalapplications, particularly as components of drug delivery devices.

The design and engineering of biomedical polymers (e.g., polymers foruse under physiological conditions) are generally subject to specificand stringent requirements. In particular, such polymeric materials mustbe compatible with the biological milieu in which they will be used,which often means that they show certain characteristics ofhydrophilicity. They also have to demonstrate adequate biodegradability(i.e., they degrade to low molecular weight species. The polymerfragments are in turn metabolized in the body or excreted, leaving notrace).

Biodegradability is typically accomplished by synthesizing or usingpolymers that have hydrolytically unstable linkages in the backbone. Themost common chemical functional groups with this characteristic areesters, anhydrides, orthoesters, and amides. Chemical hydrolysis of thehydrolytically unstable backbone is the prevailing mechanism for thedegradation of the polymer. Biodegradable polymers can be either naturalor synthetic. Synthetic polymers commonly used in medical applicationsand biomedical research include polyethyleneglycol (pharmacokinetics andimmune response modifier), polyvinyl alcohol (drug carrier), andpoly(hydroxypropylmethacrylamide) (drug carrier). In addition, naturalpolymers are also used in biomedical applications. For instance,dextran, hydroxyethylstarch, albumin and partially hydrolyzed proteinsfind use in applications ranging from plasma substitute, toradiopharmaceutical to parenteral nutrition. In general, syntheticpolymers may offer greater advantages than natural materials in thatthey can be tailored to give a wider range of properties and morepredictable lot-to-lot uniformity than can materials from naturalsources. Synthetic polymers also represent a more reliable source of rawmaterials, one free from concerns of infection or immunogenicity.Methods of preparing polymeric materials are well known in the art.However, synthetic methods that successfully lead to the preparation ofpolymeric materials that exhibit adequate biodegradability,biocompatibility, hydrophilicity and minimal toxicity for biomedical useare scarce. The restricted number and variety of biopolymers currentlyavailable attest to this.

Therefore, a need exists in the biomedical field for low-toxicity,biodegradable, biocompatible, hydrophilic polymer conjugates comprisingpharmaceutically useful modifiers, which overcome or minimize theabove-referenced problems, and which can release their drug cargo (thecorresponding drug-release product) at appropriate rates. Such polymerconjugates would find use in several applications, including componentsfor biomedical preparations, pharmaceutical formulations, medicaldevices, implants, and the packaging/delivery of therapeutic, diagnosticand prophylatic agents.

SUMMARY

In one aspect, conjugates of Formula I are described:

wherein

-   Polyal is a polyacetal or polyketal;-   Linker is a dicarboxylic acid moiety containing two or more atoms    between the carbonyls and present two or more atoms between the    carbonyl groups;-   Tether is a bifunctional organic moiety comprising a secondary or    tertiary amine and a second functional group;-   R_(a) is H, alkyl, or together with a CH₂ of the backbone of the    Tether forms a five- or six-membered ring; and-   Drug is any organic compound with a molecular weight of between    about 200 daltons and 1000 daltons, capable of covalent attachment    to the Tether and; presents the covalent attachment of Drug to    Tether via the second functional group of Tether;    wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with no reactive        hydrogen, the release half-life of Drug is from about 10 h to        more than about 300 h;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls and contains a heteroatom alpha to the        carbonyl forming the ester, the release half-life is less than        about 10 hours;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls with no heteroatom alpha to the carbonyl        forming the ester, the release half-life is more than about 100        hours;    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with a reactive        hydrogen the release half-life of Drug is from about 0.1 hours        to about 24 hours; and        wherein    -   the release half-life being measured in 0.05M phosphate buffer,        0.9% saline, pH 7.4, at 37° C.;-   with the proviso that the conjugate of Formula I is not    PHF-SA-Gly-CPT, PHF-(methyl)SA-Gly-CPT,    PHF-(2,2-dimethyl)SA-Gly-CPT, PHF-(2-nonen-2-yl)SA-Gly-CPT,    PHF-SA-Gly-Taxol, or PHF-SA-Gly-Illudin.

In another aspect, conjugates of the Formula II are described:

wherein

-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of the CH₂ is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the Drug,    or a heterocycle; or R₁ and R₂ when taken together with nitrogen to    which they are attached form a ring;-   Polyal is a polyacetal or polyketal;-   Drug is any organic compound with a molecular weight of between    about 200 daltons and 1000 daltons, capable of covalent attachment    to the Tether;    wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with no reactive        hydrogen, the release half-life of Drug is from about 10 h to        more than about 300 h;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls and contains a heteroatom alpha to the        carbonyl forming the ester, the release half-life is less than        about 10 hours;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls with no heteroatom alpha to the carbonyl        forming the ester, the release half-life is more than about 100        hours;        wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with a reactive        hydrogen, the release half-life of Drug is from about 0.1 hours        to about 20 hours;        wherein    -   the release half-life being measured in 0.05M phosphate buffer,        0.9% saline, pH 7.4, at 37° C.;        with the proviso that the conjugate is not PHF-SA-Gly-CPT,        PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT,        PHF-(2-nonen-2-yl)SA-Gly-CPT, PHF-SA-Gly-Taxol, or        PHF-SA-Gly-Illudin.

In some embodiments, the polyal is an acetal. In other embodiments, thepolyal is a ketal. In some embodiments, the acetal is PHF. In someembodiments, R₁ is H. In other embodiments, R₁ is CH₃. In someembodiments, R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains ofthe naturally occurring amino acids. In some embodiments, R₂ is an arylgroup. In some embodiments, R₂ is an heteroaryl group. In otherembodiments, R₂ is an aliphatic ring. In some embodiments, R₂ is analiphatic chain. In some embodiments, R₂ is a heterocyclic aliphaticring. In some embodiments, R₁ and R₂ when taken together with nitrogento which they are attached form a ring. In some embodiments, the ringwhich R₁ and R₂ form is a five-membered ring. In some embodiments, thering which R₁ and R₂ form is a six-membered ring. In some embodiments, Xis —CH₂—. In some embodiments, X is —OCH₂—. In some embodiments, X is—CH₂CH₂—. In some embodiments, X is optionally substituted with a C₁-C₆alkyl group. In some embodiments, Tether is selected from the groupconsisting of an amino acid, a diamine, an aminoalcohol and anaminothiol. In some embodiments, Drug is a fumagillol analog. In someembodiments, Drug is a vinca alkaloid. In some embodiments, Drug is anon-natural camptothecin. In some embodiments, the non-naturalcamptothecin is SN38. In some embodiments, the conjugate is selectedfrom the group consisting of

wherein k ranges from 1-30, m ranges from 0-300, and n ranges from100-750, and wherein the polyal comprises randomly distributedcovalently bound monomer blocks shown in brackets; and pharmaceuticallyacceptable salts thereof.

In another aspect, conjugates of the Formula III are described:

wherein

-   Polyal is a polyacetal or polyketal;-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂— is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the    —NHC(O)— of the vinca alkaloid derivative, or a heterocycle; or R₁    and R₂, when taken together with nitrogen to which they are    attached, form a ring;-   R₇ is —CH₃ or —CHO; and-   R₈ is —OCOCH₃ or OH.

In another aspect, conjugates of the formula IV are described:

whereinPolyal is a polyacetal or polyketal;

-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂— is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the —O—    of the non-natural camptothecin derivative, or a heterocycle; or R₁    and R₂ when taken together with nitrogen to which they are attached    form a ring;-   R₃ is —H, —Cl, —F, —OH or alkyl; or R₃ and R₄, may be taken together    to form a five- or six-membered ring;-   R₄ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃, or    —Si((CH₃)₂)-t-Butyl;-   R₅ is —CH₂—N(CH₃)₂, NH₂, or NO₂;-   R₆ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or    —N-4-methylcyclohexylamine;-   or R₅ and R₆, may be taken together to form a six-membered    optionally substituted ring;-   p is 0 or 1; and-   q is 0 or 1;-   with the proviso that the conjugate is not PHF-SA-Gly-CPT,    PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT, or    PHF-(2-nonen-2-yl)SA-Gly-CPT.

In another aspect, a method of identifying a Polyal-Drug conjugatehaving a drug release half-life of between about 0.1 hours and greaterthan 300 hours, as measured in phosphate buffered saline (PBS) at 37° C.is described, the method comprising: selecting a dicarboxylic acidLinker; obtaining a conjugate with said Linker, the conjugate comprisingPolyal, Drug, and said Linker; and determining the release half-life ofDrug from the conjugate.

In another aspect, pharmaceutical compositions comprising apolyal-non-natural camptothecin conjugate or a pharmaceuticallyacceptable salt of a polyal-non-natural camptothecin conjugate and apharmaceutically acceptable carrier are provided.

In another aspect, methods of treating cancer, comprising administeringto a subject in need thereof a polyal-non-natural camptothecin conjugateor a pharmaceutically acceptable salt of a polyal-non-naturalcamptothecin conjugate in an amount effective to treat the cancer aredescribed.

In some embodiments, the polyal-non-natural camptothecin useful fortreating cancer is a PHF-non-natural camptothecin conjugate. In anotherembodiment, the PHF-non-natural camptothecin conjugate useful fortreating cancer is PHF-SN38 conjugate.

In some embodiments, the cancer is selected from the group consistingof: anal, astrocytoma, leukemia, lymphoma, head and neck, liver,testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal,mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder,breast, uterus, ovary, prostate, lung, colon, pancreas, renal, andgastric.

In another aspect, pharmaceutical compositions comprising a polyal-vincaalkaloid conjugate or a pharmaceutically acceptable salt of apolyal-vinca alkaloid conjugate and a pharmaceutically acceptablecarrier are provided.

In another aspect, methods of treating cancer, comprising administeringto a subject in need thereof a polyal-vinca alkaloid conjugate or apharmaceutically acceptable salt of a polyal-vinca alkaloid conjugate inan amount effective to treat the cancer are described.

In some embodiments, the polyal-vinca alkaloid conjugate useful fortreating cancer is a PHF-vinca alkaloid conjugate.

In some embodiments, the cancer is selected from the group consistingof: anal, astrocytoma, leukemia, lymphoma, head and neck, liver,testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal,mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder,breast, uterus, ovary, prostate, lung, colon, pancreas, renal, andgastric.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the release of PHF-SA-Gly-SN38 in PBS buffer.

FIG. 2 depicts the release of PHF-SA-Gly-SN38 in human plasma.

FIG. 3 depicts the release of PHF-SA-Gly-SN38 in mouse plasma.

FIG. 4 depicts the release of PHF-GA-Gly-SN38 in PBS buffer.

FIG. 5 depicts the release of PHF-GA-Gly-SN38 in human plasma.

FIG. 6 depicts the release of PHF-GA-Gly-SN38 in mouse plasma.

FIG. 7 depicts the release of PHF-SA-Ala-SN38 in PBS buffer.

FIG. 8 depicts the release of PHF-SA-Ala-SN38 in human plasma.

FIG. 9 depicts the release of PHF-SA-Ala-SN38 in mouse plasma.

FIG. 10 depicts the release of PHF-GA-Ala-SN38 in PBS buffer.

FIG. 11 depicts the release of PHF-GA-Ala-SN38 in human plasma.

FIG. 12 depicts the release of PHF-GA-Ala-SN38 in mouse plasma.

FIG. 13 depicts the responsiveness of HCT116 tumor cells treated withPHF-non-natural camptothecin conjugates as shown in terms of percenttumor growth delay (% TGD), defined as the percent increase in mediantime to endpoint for mice treated with an agent compared to thosetreated with saline, or mean or median tumor volume, for mice treatedwith an agent compared to those treated with saline.

DETAILED DESCRIPTION

Definitions

The following definitions are used in connection with the Polyal-Drugconjugates comprising variable rate-releasing linkers:

“Alkyl” refers to a hydrocarbon chain that may be a straight chain orbranched chain. The chain my contain an indicated number of carbonatoms. For example, C₁-C₆ indicates that the group may have from 1 to 6(inclusive) carbon atoms in it.

“Aryl” refers to cyclic aromatic carbon ring systems containing from 6to 18 carbons. Examples of an aryl group include, but are not limitedto, phenyl, naphthyl, anthracenyl, tetracenyl, and phenanthrenyl. Anaryl group can be unsubstituted or substituted with one or more of thefollowing groups: H, halogen, CN, OH, aryl, arylalkyl, heteroaryl,heteroarylalkyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₃fluorinated-alkyl, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyl-C₁₋₃alkyl, NO₂, NH₂,NHC₁₋₆ alkyl, N(C₁₋₆ alkyl)₂, NHC₃₋₆ cycloalkyl, N(C₃₋₆ cycloalkyl)₂,NHC(O)C₁₋₆ alkyl, NHC(O)C₃₋₆ cycloalkyl, NHC(O)NHC₁₋₆ alkyl,NHC(O)NHC₃₋₆ cycloalkyl, SO₂NH₂, SO₂NHC₁₋₆ alkyl, SO₂NHC₃₋₆ cycloalkyl,SO₂N(C₁₋₆ alkyl)₂, SO₂N(C₃₋₆ cycloalkyl)₂, NHSO₂C₁₋₆ alkyl, NHSO₂C₃₋₆cycloalkyl, CO₂C₁₋₆ alkyl, CO₂C₃₋₆ cycloalkyl, CONHC₁₋₆ alkyl, CONHC₃₋₆cycloalkyl, CON(C₁₋₆ alkyl)₂, CON(C₃₋₆ cycloalkyl)₂OH, OC₁₋₃ alkyl, C₁₋₃fluorinated-alkyl, OC₃₋₆ cycloalkyl, OC₃₋₆ cycloalkyl-C₁₋₃ alkyl, SH,SO_(x)C₁₋₃ alkyl, C₃₋₆ cycloalkyl, or SO_(x)C₃₋₆ cycloalkyl-C₁₋₃ alkyl,where x is 0, 1, or 2.

“Heteroaryl” refers to mono and bicyclic aromatic groups of 4 to 10atoms containing at least one heteroatom. Heteroatom as used in the termheteroaryl refers to oxygen, sulfur and nitrogen. Examples of monocyclicheteroaryls include, but are not limited to, oxazinyl, thiazinyl,diazinyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl, isoxazolyl,furanyl, furazanyl, oxazolyl, thiazolyl, thiophenyl, pyrazolyl,triazolyl, and pyrimidinyl. Examples of bicyclic heteroaryls include butare not limited to, benzimidazolyl, indolyl, isoquinolinyl, indazolyl,quinolinyl, quinazolinyl, purinyl, benzisoxazolyl, benzoxazolyl,benzthiazolyl, benzodiazolyl, benzotriazolyl, isoindolyl and indazolyl.A heteroaryl group can be unsubstituted or substituted with one or moreof the following groups: H, halogen, CN, OH, aryl, arylalkyl,heteroaryl, heteroarylalkyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₁₋₃ fluorinated-alkyl, C₃₋₆ cycloalkyl, C₃₋₆cycloalkyl-C₁₋₃alkyl, NO₂,NH₂, NHC₁₋₆ alkyl, N(C₁₋₆ alkyl)₂, NHC₃₋₆ cycloalkyl, N(C₃₋₆cycloalkyl)₂, NHC(O)C₁₋₆ alkyl, NHC(O)C₃₋₆ cycloalkyl, NHC(O)NHC₁₋₆alkyl, NHC(O)NHC₃₋₆ cycloalkyl, SO₂NH₂, SO₂NHC₁₋₆ alkyl, SO₂NHC₃₋₆cycloalkyl, SO₂N(C₁₋₆ alkyl)₂, SO₂N(C₃₋₆ cycloalkyl)₂, NHSO₂C₁₋₆ alkyl,NHSO₂C₃₋₆ cycloalkyl, CO₂C₁₋₆ alkyl, CO₂C₃₋₆ cycloalkyl, CONHC₁₋₆ alkyl,CONHC₃₋₆ cycloalkyl, CON(C₁₋₆ alkyl)₂, CON(C₃₋₆ cycloalkyl)₂OH, OC₁₋₃alkyl, C₁₋₃ fluorinated-alkyl, OC₃₋₆ cycloalkyl, OC₃₋₆ cycloalkyl-C₁₋₃alkyl, SH, SO_(x)C₁₋₃ alkyl, C₃₋₆ cycloalkyl, or SO_(x)C₃₋₆cycloalkyl-C₁₋₃ alkyl, where x is 0, 1, or 2.

“C₁-C₆ alkyl” refers to a straight or branched chain saturatedhydrocarbon containing 1-6 carbon atoms. Examples of a C₁-C₆ alkyl groupinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-pentyl, isopentyl, neopentyl, and hexyl.

“C₁-C₆ alkoxy” refers to a straight or branched chain saturated orunsaturated hydrocarbon containing 1-6 carbon atoms and at least oneoxygen atom. Examples of a C₁-C₆-alkoxy include, but are not limited to,methoxy, ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy,and hexoxy.

“Cycloalkyl” refers to a cyclic saturated hydrocarbon. Examples of acycloalkyl group include, but are not limited to, cyclopropane,cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane.

“Heterocycle” refers to a cyclic saturated hydrocarbon wherein at leastone of the carbons is replaced by N, S, or O. Examples of heterocycleinclude, but are not limited to, azetidine, oxetane, thietane,azolidine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene,imidazolidine, oxazolidine, thiazolidine, morpholine, piperidine,tetrahydropyran, thiane, piperazine, oxazine, and dioxane.

“Halogen” refers to an atom of fluorine, chlorine, bromine, or iodine.

“Cyclized imide” and “cyclic-imide” refer to either saturated orunsaturated cyclic or heterocyclic compounds that contain the imidefunctional group which consists of two carbonyl groups bound to anitrogen atom. Cyclic-imides can be further substituted with otherfunctional groups. Examples of a cyclic-imide include, but are notlimited to, piperidyl-2,6-dione, morpholyl-3,5-dione, andpyrrolidyl-2,5-dione.

The term “optionally substituted CH₂” when used herein means that one orboth hydrogen atoms may be substituted with one or more of the followinggroups: OH, halogen, CN, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ alkoxy, C₁-C₃ fluorinated alkyl, NO₂, NH₂, NHC₁-C₆ alkyl, N(C₁-C₆alkyl)₂, NHC(O)C₁-C₆ alkyl, NHC(O)NHC₁-C₆ alkyl, SO₂NH₂, SO₂NHC₁-C₆alkyl, SO₂N(C₁-C₆ alkyl)₂, NHSO₂C₁-C₆ alkyl, C(O)OC₁-C₆ alkyl, CONHC₁-C₆alkyl, CON(C₁-C₆ alkyl)₂, C₁-C₆ alkyl, or both hydrogen atoms may besubstituted and the substituted groups when taken together with thecarbon to which they are attached, form a cycloalkyl orheterocycloalkyl, each optionally substituted with C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, CO₂C₁-C₆ alkyl, CN, OH,cycloalkyl, CONH₂, aryl, heteroaryl, COaryl, or trifluoroacetyl.

The term “pharmaceutically acceptable salts” include, e.g.,water-soluble and water-insoluble salts, such as the acetate, amsonate(4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calciumedetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate,einbonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts.

The term “PHF” means [poly-(1-hydroxymethylethylene hydroxy-methylformal)].

The term “non-natural camptothecin” means a compound based on thestructure of the natural product camptothecin (CPT). Non-limitingexamples of non-natural camptothecins include topotecan, SN-38,9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan,lurtotecan, exatecan, diflomotecan, belotecan, and S39625.

The term “fumagillol analog” means any fumagillin core structure,including fumagillamine, that inhibits the ability of MetAP-2 to removeNH₂-terminal methionines from proteins as described in Rodeschini etal., J. Org. Chem., 69, 357-373, 2004 and Liu, et al., Science 282,1324-1327, 1998. Nonlimiting examples of “fumagillol analogs” aredisclosed in J. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870,2005; European Patent Application 0 354 787; J. Med. Chem., 49, 5645,2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91,2004; Tet. Lett. 40, 4797, 1999; WO99/61432; U.S. Pat. Nos. 6,603,812;5,789,405; 5,767,293; 6,566,541; and 6,207,704.

The term “polyal” means a polymer having at least one acetal or ketaloxygen atom in each monomer unit positioned within the main chain.Examples of polyals can be found in U.S. Pat. Nos. 5,811,510, 5,863,990,5,958,398 and international application PCT/US2004/029130 which areincorporated herein by reference in their entirety. In certainembodiments, biodegradable biocompatible polymer carriers, useful forpreparation of polymer conjugates described herein, are naturallyoccurring polysaccharides, glycopolysaccharides, and synthetic polymersof polyglycoside, polyacetal, polyamide, polyether, and polyester originand products of their oxidation, functionalization, modification,cross-linking, and conjugation. When the monomer units of a polyal aredepicted herein the two free hydroxyls therein are equally reactiveduring derivatization and therefore either hydroxyl may be actuallyderivatized not just the one depicted.

The following abbreviations are used herein and have the indicateddefinitions: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride), ACN (acetonitrile), CPT (camptothecin), Gly (glycine),Ala (alanine), DMAP (dimethylamino pyridine), PHF-GA(poly(1-hydroxymethylethylene hydroxymethyl-formal) conjugated toglutaric acid), PHF-SA (poly(1-hydroxymethylethylenehydroxymethyl-formal) conjugated to succinic acid), DMF (dimethylformamide), HPLC (high pressure liquid chromatography), TBDPS(tert-butyldiphenylsilyl), TBAF (Tetra-n-butylammonium fluoride), FBS(fetal bovine serum), PBS (phosphate buffered saline (0.05M phosphate,0.9% saline)), DCM (dichloromethane), DIPC (diisopropylcarbodiimide), DI(deionized), RP (reverse-phase), SEC (size exclusion), r.t. (roomtemperature).

Variable Rate-Releasing Linkers

In has been unexpectedly discovered that amidoester linkages utilized tolink polymeric carriers with drugs are capable of releasing the drugs orprodrugs under physiological conditions, in a pH-dependent manner. Suchlinkages comprise a dicarboxylic acid attached to a hydroxyl moiety of apolyhydroxylated polymer carrier such as, for example, a polyal, via anester bond, and an amino group containing a bifunctional tether via anamide bond. Tether can provide for functional modification of Drug inorder to introduce the amino group which is capable of forming the amidewith the dicarboxylic acid moiety in the process of drug conjugation.

In one aspect of this disclosure, conjugates of the Formula I aredescribed

wherein

-   Polyal is a polyacetal or polyketal; C-b1-   Linker is a dicarboxylic acid moiety containing two or more atoms    between the carbonyls; C-b3-   Tether is a bifunctional organic moiety comprising a secondary or    tertiary amine;-   R_(a) is H, alkyl, or together with a CH₂ of the backbone of the    Tether forms a five- or six-membered ring; and C-b2-   Drug is any organic compound with a molecular weight of between    about 200 daltons and 1000 daltons, capable of covalent attachment    to the Tether;    wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with no reactive        hydrogen, the release half-life of Drug is from about 10 h to        more than about 300 h;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls and contains a heteroatom alpha to the        carbonyl forming the ester, the release half-life is less than        about 10 hours;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls with no heteroatom alpha to the carbonyl        forming the ester, the release half-life is more than about 100        hours;        wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with a reactive        hydrogen the release half-life of Drug is from about 0.1 hours        to about 24 hours;        wherein    -   the release half-life being measured in 0.05M phosphate buffer,        0.9% saline, pH 7.4, at 37° C.;        with the proviso that the compound is not PHF-SA-Gly-CPT,        PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT,        PHF-(2-nonen-2-yl)SA-Gly-CPT, PHF-SA-Gly-Taxol, or        PHF-SA-Gly-Illudin.

In another aspect, conjugates of the Formula II are described:

wherein

-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of the CH₂ is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the Drug,    or a heterocycle; or R₁ and R₂ when taken together with nitrogen to    which they are attached form a ring;-   Polyal is a polyacetal or polyketal; and-   Drug is any organic compound with a molecular weight of between    about 200 daltons and 1000 daltons, capable of covalent attachment    to the Tether;    wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with no reactive        hydrogen, the release half-life of Drug is from about 10 h to        more than about 300 h;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls and contains a heteroatom alpha to the        carbonyl forming the ester, the release half-life is less than        about 10 hours;    -   when Linker is a dicarboxylic acid with at least three atoms        between the carbonyls with no heteroatom alpha to the carbonyl        forming the ester, the release half-life is more than about 100        hours;        wherein    -   when Linker is a dicarboxylic acid with two atoms between the        carbonyls and Tether contains a nitrogen with a reactive        hydrogen the release half-life of Drug is from about 0.1 hours        to about 24 hours;        wherein    -   the release half-life being measured in 0.05M phosphate buffer,        0.9% saline, pH 7.4, at 37° C.;        with the proviso that the compound is not PHF-SA-Gly-CPT,        PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT,        PHF-(2-nonen-2-yl)SA-Gly-CPT, PHF-SA-Gly-Taxol, or        PHF-SA-Gly-Illudin.

In some embodiments, polyal is an acetal.

In some embodiments, polyal is a ketal.

In some embodiments, the acetal is PHF.

In some embodiments, R₁ is H.

In some embodiments, R₁ is CH₃.

In some embodiments, R₂ is —CH(Y)—C(O)—, wherein Y is one of the sidechains of the naturally occurring amino acids.

In some embodiments, R₂ is an aromatic group.

In some embodiments, R₂ is a heteroaryl group.

In some embodiments, R₂ is an aliphatic group.

In some embodiments, R₂ is an aliphatic chain.

In some embodiments, R₂ is a heterocyclic aliphatic ring.

In some embodiments, R₁ and R₂, when taken together with nitrogen towhich they are attached, form a ring.

In some embodiments, the ring which R₁ and R₂ form is a five-memberedring.

In some embodiments, the ring which R₁ and R₂ form is a six-memberedring.

In some embodiments, X is —CH₂—.

In some embodiments, X is —OCH₂—.

In some embodiments, X is —CH₂CH₂—.

In some embodiments, X is optionally substituted with a C₁-C₆ alkylgroup.

In some embodiments, the bifunctional tether —(N—R₂)— is an amino acid,a diamine, an aminoalcohol or an aminothiol.

In some embodiments, Drug is fumagillol.

In some embodiments, Drug is a vinca alkaloid.

In some embodiments, Drug is a non-natural camptothecin.

In another aspect, a method of identifying a Polyal-Drug conjugatehaving a drug release half-life of between about 0.1 hours and greaterthan 300 hours as measured in PBS buffer at 37° C. is described, themethod comprising: selecting a dicarboxylic acid Linker; obtaining aconjugate with said Linker, the conjugate comprising Polyal, Drug, andsaid Linker; and determining the release half-life of Drug from theconjugate.

It has been discovered that by judicious choice of Linker and Tether,Drug can be released from the Polyal-Drug conjugate via at least twoindependent pathways, either (A) intramolecular rearrangement of theamidoester linkage resulting in cleavage of the Polyal-ester bondaccompanied by formation of a cyclic imide at the Drug site; or (B)hydrolysis of the ester bond between Polyal and the amidoester Linkerresulting in release of Drug-amidoacid derivative (see Scheme 1).

When R₁ is H and X is CH₂, the cleavage of Drug from Polyal proceedsthrough process A. Under most other conditions, the ester portion of thelinkage undergoes hydrolysis.

The stability of the amidoester linkages is pH-dependent with anincrease in the pH of an aqueous solution accelerating bothintramolecular rearrangement of the amidoester and ester bond hydrolysisof the Polyal-Drug conjugate. When the Polyal-Drug conjugate isevaluated at physiological conditions, i.e., 0.05M phosphate pH 7.4buffered saline, 0.9% NaCl (PBS), at 37° C., the predominant mechanism(process A (intramolecular) or process B (intermolecular hydrolysis))and the rate of release of Drug from Polyal can be influenced bystructural characteristics of the amidoester based on careful selectionof the dicarboxylic acid Linker and the amine-containing Tether attachedto Drug.

Intramolecular Release (Process A)

For Linkers with two atoms between the carbonyl groups of thedicarboxylic acid linker (e.g. succinic acid derivatives (SA)) therelease product composition and the rate of release may be effectivelycontrolled by a combination of steric and electronic effects in both thedicarboxylic acid Linker and the amine-containing Tether. For succinicacid derivatives, the release half-life of Drug (in PBS buffer, pH 7.4,at 37° C.) can be adjusted to between from about 0.1 h to greater than100 h. The release product composition can vary from predominantly thecyclic succinimide drug derivatives which result from the intramolecularrelease process, to succinic acid amide drug derivatives, which resultfrom an intermolecular release process. Both of these processes dependupon the selection of the amine-containing Tether employed.

For example, when the release of succinimide derivatives is desired(i.e. intramolecular release process A) the release half-life ofPolyal-Drug conjugate can be adjusted by altering the steric effect ofthe R₂ group. Increasing this steric effect hinders the intramolecularnucleophilic attack of the nitrogen on the carbonyl of the ester end ofthe linkage. Conjugates V1, V2, and V3 (Scheme 2) for which theamine-containing tether is glycine, β-alanine and alanine respectively,the increase in the steric hindrance of Tether at R₂(glycine<β-alanine<alanine) results in an increase of the releasehalf-life of Drug from 3.4 hours to 17 hours and 19 hours, respectively,when tested in PBS, pH 7.4 at 37° C.

The release half-life can be attenuated also by the electronicproperties of the R₂ moiety. For example, when R₂ is an aromatic ringbound to the nitrogen, substituents on the aromatic ring that influencethe electronic density on the nitrogen affect the rate of nucleophilicattack of the nitrogen on the carbonyl.

Intermolecular Release (Process B)

When the ester portion of the amidoester linkage is targeted as theprimary conjugate drug release product (i.e. by intermolecular releasemechanism), the release half-life of Drug can be adjusted by employingLinkers with differing numbers of atoms between the two carbonyls of thedicarboxylic acid, the electronic influence alpha to the carbonylforming the ester (e.g. glutaric acid (GA) and oxaglutaric acid (OGA)),and Linker/Tether combinations (e.g. succinic acid-Tether derivatives).

For succinic acid derivatives, the release half-life of thecorresponding succinic acid amide derivatives from the conjugates can beattenuated by changing the amine-containing Tethers. Use of secondaryamine tethers (i.e. amines in which the nitrogen does not have areactive hydrogen directly attached to the nitrogen) can be used toeliminate the possibility of succinimide formation (i.e. intramolecularrelease process), and the steric hindrance at R₂ will control therelease half-life of Drug via the intermolecular mechanism. For example,the Tether sarcosine along with succinic acid provides a releasehalf-life of Drug of 81 hours for the conjugate V8, while conjugate V13,with the more hindered Tether proline, instead of sarcosine, under thesame conditions, (PBS, pH 7.4, 37° C.) releases Drug with a half-life of375 hours.

Linkers with three or more atoms in the carbon or heteroatom chainconnecting the carboxyl groups in the dicarboxylic acid linker degradeprimarily via ester bond hydrolysis via an intermolecular mechanism anddirectly release Drug-amidoacid derivatives (pathway B, Scheme 1). Forexample, conjugates utilizing the Linker glutaric acid exhibit releasehalf-lives of Drug of more than 100 hours in PBS pH 7.4 at 37° C. Theseare known as extended release Linkers.

Another example of Linkers with three or more atoms between the twocarbonyls of the dicarboxylic acid is oxaglutaric acid (OGA). Conjugatesutilizing the Linker oxaglutaric acid release through the intermolecularprocess and exhibit release half-lives of Drug of less than 10 hours(PBS, pH 7.4, 37° C.). OGA is characterized as a fast release Linker. Byway of explanation and without intending to be bound to any particulartheory, the oxygen atom in the OGA group, which is in an alpha positionto the carbonyl forming the ester, has an electron-withdrawing effect onthe ester bond between PHF and OGA, thus making it more susceptible tohydrolysis.

Tethers:

Tethers are bifunctional organic moieties of between about 50 daltonsand about 300 daltons, comprising a secondary or tertiary amine.Bifunctional organic moieties are straight, branched or cyclic aliphaticalkyl groups comprising at least one heteroatom selected from N, O, andS, in addition to the secondary or tertiary amine, and NH₂-aryl andNH₂-heteroaryl groups substituted with at least one heteroatom selectedfrom N, O, and S, the alkyl group optionally containing aryl groups.Nonlimiting examples of various tethers are listed below:

1. Amino acids

2. Aryl and Heteroaryl Groups

3. Heterocycles

4. Alkyl groups

5. Cycloalkyl Groups

Illustrative non-limiting examples of Polyal-Drug conjugates employingvarious variable rate-releasing linkers are listed below:

Polyal-Fumagillol Conjugates

Polyal-Vinca Conjugates

Compounds of the Formula III are described:

wherein

-   Polyal is a polyacetal or polyketal;-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂— is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the    —NHC(O)— of the vinca alkaloid derivative, or a heterocycle; or R₁    and R₂, when taken together with nitrogen to which they are    attached, form a ring;-   R₇ is —CH₃ or —CHO; and-   R₈ is —OCOCH₃ or OH.

Illustrative non-limiting examples of compounds of Formula III arelisted below:

Polyal-Non-natural Camptothecin Conjugates

Compounds of the formula IV are described:

wherein

-   Polyal is a polyacetal or polyketal;-   X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂— is    optionally substituted;-   R₁ is H or CH₃;-   R₂ is —CH(Y)—C(O)—, wherein Y is one of the side chains of the    naturally occurring amino acids, an aryl group, a heteroaryl group,    a cycloalkyl, an alkyl group attached to both the N—R₁ and the —O—    of the non-natural camptothecin derivative, or a heterocycle; or R₁    and R₂ when taken together with nitrogen to which they are attached    form a ring;-   R₃ is —H, —Cl, —F, —OH or alkyl; or R₃ and R₄, may be taken together    to form a five- or six-membered ring;-   R₄ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃, or    —Si((CH₃)₂)-t-Butyl;-   R₅ is —CH₂—N(CH₃)₂, NH₂, or NO₂;-   R₆ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or    —N-4-methylcyclohexylamine;-   or R₅ and R₆, may be taken together to form a six-membered    optionally substituted ring;-   p is 0 or 1; and-   q is 0 or 1;-   with the proviso that the compound is not PHF-SA-Gly-CPT,    PHF-(methyl)SA-Gly-CPT, PHF-(2,2-dimethyl)SA-Gly-CPT, or    PHF-(2-nonen-2-yl)SA-Gly-CPT.

Illustrative non-limiting examples of compounds of Formula IV are listedbelow:

PHF-SN38 Conjugates

wherein k ranges from 1-30, m ranges from 0-300, and n ranges from100-750, and wherein the polyal comprises randomly distributedcovalently bound monomer blocks shown in brackets; and pharmaceuticallyacceptable salts thereof.Methods of Using the Polyal-Drug Conjugates Comprising VariableRate-Releasing Linkers

The rate of release of a drug from a polymeric conjugate can play a verysignificant role in altering the properties of the released drug,including having effects on the overall efficacy of the released drug,the duration of action of the released drug, the frequency of dosingrequired, the toxicity of the released drug, the biodistribution of thereleased drug, and the overall pharmacokinetic and pharmacodynamicproperties of the released drug. For example, a slow, continuous releaseof a drug from a polymeric conjugate can mimic the effect of a slow,continuous infusion of the drug. Such a delivery can be beneficial, forexample, with a drug-release product which has an inherently short-halflife, and therefore would require much more frequent dosing ifadministered directly, without the benefit of conjugation to a polymer.Furthermore, a polymer conjugate of a drug release product could bedesigned to alter the C_(max) of a drug-release product. By carefullydesigning a polymer conjugate with an appropriate release half-life, aC_(max) value can be targeted such that it falls within a desiredtherapeutic window. For example, a C_(max) value lower than a valueknown to have an associated toxicity for a known drug, while maintainingan exposure level known to be a therapeutic level of the drug-releaseproduct.

In another aspect, compositions comprising polyal-non-naturalcamptothecin conjugates or a pharmaceutically acceptable salt of apolyal-non-natural camptothecin conjugate and a pharmaceuticallyacceptable carrier are provided.

In another aspect, methods of treating cancer, comprising administeringto a subject in need thereof a polyal-non-natural camptothecin conjugateor a pharmaceutically acceptable salt of a polyal-non-naturalcamptothecin conjugate in an amount effective to treat the cancer aredescribed.

In some embodiments, the polyal-non-natural camptothecin useful fortreating cancer is a PHF-non-natural camptothecin conjugate. In otherembodiments, the PHF-non-natural camptothecin conjugate useful fortreating cancer is PHF-SN38 conjugate.

In some embodiments, the cancer is selected from the group consistingof: anal, astrocytoma, leukemia, lymphoma, head and neck, liver,testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal,mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder,breast, uterus, ovary, prostate, lung, colon, pancreas, renal, andgastric.

Therapeutic Administration of the Polyal-Non-Natural CamptothecinConjugates

When administered to a subject, the polyal-non-natural camptothecinconjugates or pharmaceutically acceptable salts of thepolyal-non-natural camptothecin conjugates can be administered as acomponent of a composition that comprises a physiologically acceptablecarrier or vehicle. The compositions described herein can be preparedusing a method comprising admixing the polyal-non-natural camptothecinconjugates or a pharmaceutically acceptable salt of thepolyal-non-natural camptothecin conjugates and a physiologicallyacceptable carrier, excipient, or diluent. Admixing can be accomplishedusing methods well known for admixing a polyal-non-natural camptothecinconjugates or a pharmaceutically acceptable salt of thepolyal-non-natural camptothecin conjugates and a physiologicallyacceptable carrier, excipients, or diluents.

The polyal-non-natural camptothecin conjugates or pharmaceuticallyacceptable salts of polyal-non-natural camptothecin conjugates can beadministered by any convenient route, for example, by infusion or bolusinjection and can be administered together with another therapeuticagent. Administration of the polyal-non-natural camptothecin conjugatewill result in release of a non-natural camptothecin into thebloodstream.

In one embodiment, the polyal-non-natural camptothecin conjugate or apharmaceutically acceptable salt of the polyal-non-natural camptothecinconjugate is administered intravenously.

In another aspect, compositions comprising polyal-vinca alkaloidconjugates or a pharmaceutically acceptable salt of a polyal-vincaalkaloid conjugate and a pharmaceutically acceptable carrier areprovided.

In another aspect, methods of treating cancer, comprising administeringto a subject in need thereof a polyal-vinca alkaloid conjugate or apharmaceutically acceptable salt of a polyal-vinca alkaloid conjugate inan amount effective to treat the cancer are described.

In some embodiments, the polyal-vinca alkaloid conjugate useful fortreating cancer is a PHF-vinca alkaloid conjugate.

In some embodiments, the cancer is selected from the group consistingof: anal, astrocytoma, leukemia, lymphoma, head and neck, liver,testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal,mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder,breast, uterus, ovary, prostate, lung, colon, pancreas, renal, andgastric.

Therapeutic Administration of the Polyal-Vinca Alkaloid Conjugates

When administered to a subject, the polyal-vinca alkaloid conjugates orpharmaceutically acceptable salts of the polyal-vinca alkaloidconjugates can be administered as a component of a composition thatcomprises a physiologically acceptable carrier or vehicle. Thecompositions described herein can be prepared using a method comprisingadmixing the polyal-vinca alkaloid conjugates or a pharmaceuticallyacceptable salt of the polyal-vinca alkaloid conjugates and aphysiologically acceptable carrier, excipient, or diluent. Admixing canbe accomplished using methods well known for admixing a polyal-vincaalkaloid conjugates or a pharmaceutically acceptable salt of thepolyal-vinca alkaloid conjugates and a physiologically acceptablecarrier, excipients, or diluents.

The polyal-vinca alkaloid conjugates or pharmaceutically acceptablesalts of polyal-vinca alkaloid conjugates can be administered by anyconvenient route, for example, by infusion or bolus injection and can beadministered together with another therapeutic agent. Administration ofthe polyal-vinca alkaloid conjugate will result in release of a vincaalkaloid into the bloodstream.

In one embodiment, the polyal-vinca alkaloid conjugate or apharmaceutically acceptable salt of the polyal-vinca alkaloid conjugateis administered intravenously.

Methods of Making Various Polyal-Drug Conjugates Comprising VariableRate-Releasing Linkers

The polyal-drug conjugates comprising variable rate-releasing linkersand their pharmaceutically acceptable salts can be prepared using avariety of methods starting from commercially available compounds, knowncompounds, or compounds prepared by known methods. The polyal-drugconjugates comprising variable rate-releasing linkers can be preparedusing a variety of methods starting from commercially availablecompounds, known compounds, or compounds prepared by known methods.General synthetic routes to many of the compounds described are includedin the following schemes. It is understood by those skilled in the artthat protection and deprotection steps not shown in the Schemes may berequired for these syntheses, and that the order of steps may be changedto accommodate functionality in the target molecule.

Methods useful for making the polyal-drug conjugates comprising variablerate-releasing linkers are set forth in the Examples below andgeneralized in the following schemes.

Reaction of the C₂₃ ester of a vinca alkaloid with hydrazine followed bytreatment of the resulting product with NaNO₂ results in an active azidoester. Reaction of the azido ester with an amino tether such asethanolamine or propanolamine results in a vinca alkaloid derivativewith a functionalized hydroxyl which can be further derivatized withamino containing tethers for conjugation to a polyal through adicarboxylic acid (see Scheme 2).

wherein m and n are 0-300, and 100-750, respectively.

Treatment of the hydroxyl derivative of the vinca alkaloid with aprotected amino containing tether such as t-butoxy esterified amino acidfollowed by TFA hydrolysis of the ester affords the triflate salt of thevinca alkaloid. Conjugation of the vinca alkaloid to the polyalsderivatized previously with a dicarboxylic acid, as reported in U.S.2007/019008, is effected with the use of an activating agent such as EDCin water/acetonitrile as solvent. After completion, DI water was addedso that the ACN is less than 10% of total the volume and the product waspurified by gel filtration column (Sephadex G-25, water as eluent).

The 10-hydroxy group of non-natural camptothecin derivative, forexample, SN38, is selectively protected by reacting the derivative withtert-butyldiphenylsilyl chloride in the presence of triethylamine.Subsequent glycination of the 20-hydroxy group by reacting witht-butylcarbonyl-glycine to form the glycinate of the derivative isprepared according to Sapra, P. et al., Clin. Cancer Res., 14: 1888-1896(2008). Alternatively, other amino acids can be employed, e.g. alanine,which slows the release half-life from the polyal. The primary amine isunmasked by removing the Boc protecting group by treatment withtrifluoroacetic acid, followed by removing the TBDPS protecting groupwith tetrabutylammonium fluoride (see Scheme 6 below).

wherein k, m, and n are integers between 1-30, 0-300, and 100-750,respectively.

The resulting non-natural camptothecin-Gly derivative is then coupledwith the polyal PHF activated with a dicarboxylic acid such as SA, GA,or OGA, to form the desired polyal-non-natural camptothecin conjugatePHF-SN38.

PHF-Fumagillol Conjugates

The methods for making various polyal-fumagillol conjugates whichcomprise variable rate-releasing linkers can be found in U.S. Ser. No.12/276,856, the contents of which are hereby incorporated in itsentirety.

EXAMPLES Example 1 PHF-Vinca Conjugate (Conjugate V4)

Hydroxylpropylvindesine-alanine-BOC

Hydroxylpropylvindesine (0.227 g, 0.280 mmol), prepared according to themethod of Conrad et al., J. Med. Chem. 22, 391, (1979), Boc-alanine(0.058 g, 0.308 mmol) and DMAP (3.42 mg, 0.028 mmol) were dissolved in 5mL of anhydrous DCM (5 ml) and cooled to 0° C. DIPC (0.056 ml, 0.363mmol) was then added and stirred at 0° C. for 3 hours. Afterwards, thereaction mixture was washed with conc. NaHCO₃ solution and water, driedwith Na₂SO₄, filtered and finally concentrated. The crude product wasadded to a silica gel column and eluted with an ethyl acetatetriethylamine gradient (A. ethyl acetate, B1% triethylamine inmethanol,—the gradient: 100% A for 3 minutes, 0-40% B in 10 minutes)(combiflash system, 40 g silica column).

Hydroxylpropylvindesine-alanine-TFA

Hydroxylpropylvindesine-alanine-BOC (0.227 g, 0.231 mmol) was dissolvedin 2 mL 50/50 DCM/TFA, stirred at room temperature for 2 hours.Afterwards, diethylether was added to the solution to precipitate theproduct. The product was collected by centrifuge and the solventdecanted.

Conjugate V4

PHF-SA (1.600 g, 6.05 mmol) was dissolved in 20 mL DI water and 4 mL ACNand cooled to 0° C. Hydroxylpropylvindesine-alanine-TFA (0.21 g, 0.242mmol) was dissolved in 2 mL ACN and added to the solution. The pH wasadjusted to about 6 and then EDC (0.116 g, 0.605 mmol) was added. Thesolution was stirred at 0° C. for 30 minutes and then warm to r.t. Theprogress of reaction was monitored by HPLC (both SEC and RP). Aftercompletion, DI water was added so that the ACN is less than 10% of totalthe volume and the product was purified by gel filtration column(Sephadex G-25, water as eluent).

Example 2 Method for the Determination of Drug Release from PHF-DrugConjugates In Vitro

The evaluation of the process of drug release from PHF-drug conjugateswas carried out in physiological conditions in vitro. The testing wasperformed in phosphate buffered saline (0.05M phosphate pH 7.4, NaCl0.9%) at 37° C., typically over a 24-hour period. The concentration ofconjugated drug was monitored by high pressure size exclusionchromatography and the concentration of drug release product(s) wasmonitored by reverse phase HPLC using specrtophotometric detection at awavelength specific for the drug conjugate, the drug release product, ora combination of both. The drug conjugate linker degradation rates (K,h⁻¹) were estimated by linear regression analysis of the conjugated drugsemilogarithmic concentration decay profiles. Linker stability wasreported as drug release half-lives (t ½, h, where t½ was calculated asLn(2)/K).

TABLE 1 Release Half-lives of Various PHF-Vinca Alkaloid Drug Conjugates## Vinca Alkaloid Conjugate T 1/2, h V1 

3.4 V2 

17 V3 

19 V4 

24 V5 

26 V6 

65 V7 

75 V8 

81 V9 

200 V10

250 V11

360 V12

500 V13

375 V14

Vinca represents a vinca alkaloid attached to the NH through thecarboxylic acid at position C-23 of the vinca alkaloid.

TABLE 2 Release Half-lives of Various PHF-Fumagillol Analog ConjugatesFumagillol Analog Conjugates T_(1/2) h F1 

0.6 F2 

1.0 F3 

1.2 F4 

1.2 F5 

1.4 F6 

1-4 F7 

5.5 F8 

5.5 F9 

7.5 F10

8.1 F11

8.1 F12

9.1 F13

9.5 F14

7-10 F15

 20 F16

100 F17

>100  F18

500 F19

>>200  F20

>100  F21

>100  F22

>100  F23

9.1

FUM means

TABLE 3 Release Half-lives of Various SN38 Conjugates ## SN38 ConjugateT½, h S1 PHF-SA-Gly-SN38 2.0 S2 PHF-GA-Gly-SN38 18.5 S3 PHF-SA-Ala-SN3836.9 S4 PHF-GA-Ala-SN38 54.2Method for the Determination of Drug Release from PHF-Drug Conjugates InVitro

Plasma incubation was carried out in buffered plasma from mouse orhuman. Plasma was buffered to pH 7.4 with 0.5M Phosphate buffer pH 7.2at a ratio of 5:1 (v/v, plasma:buffer). A mixture of the PHF-drugconjugate in plasma was prepared at 0.8 mg/ml, aliquoted into 50 μlsamples in microcentrifuge vials and samples were transferred to a waterbath at 37° C. At time points 0, 20, 40, 60, 80, 100, 120, 140, 160,180, 200, 220 and 240 minutes, aliquots were removed at the time pointand extracted with 200 μl acetonitrile and analyzed by RP-HPLC/MS(Column Gemini C18, 150×2.0 mm, 3 μm, operating at room temperature,with an LC flow of 350 μl/min; mobile phases were: 0.1% formic acid inwater (A), and 0.1% formic acid in acetonitrile, linear gradient was10-50% B in 9.5 minutes, 50-90% B in 0.5 min, held at 90% B for 1minute, re-equilibrated for 5 minutes at 10% B). UV integration at 365nm of all the release products was performed after confirmation of therelease products by MS. Data is shown as individual UV area peaks of therelease products and the sum of them all in FIGS. 2-3, 5-6, 8-9, and11-12.

Identification of Release Products In Vitro by Mass Spectrometry inBuffer and Plasma

Release products of PHF-SN38 in PBS buffer and in human and mouse plasmawere identified by mass spectrometry in acetonitrile extracts afterprecipitation of the polymer with acetonitrile in incubation media asdescribed above. Results are shown in FIGS. 1-12.

Example 3 Inhibitory Effect of Polyal-SN38 Conjugates and Analogs onCell Growth

Using HT-29 cells, the effect of the polyal-SN38 conjugates on cellgrowth was evaluated.

Cells are grown in McCoy's 5a medium with 1.5 mM L-glutaminesupplemented with 10% FBS. The (exponentially growing) cells are seededin 24-well culture plates (about 10000 cells/well), cultured for 24hours, and then treated with test compounds at various dilutions. Growthinhibition is assessed 72 hours post treatment (MTT assay). The resultsare shown in Table 4.

TABLE 4 HT29 HCT116 Compound IC50 (uM) IC50 (uM) SN38-ALA-GA 0.121 0.024SN38-ALA-SI 0.457 0.12 SN38-ALA-SA 0.187 0.055 SN38-GLY-SA 0.117 0.032SN38-GLY-SI 0.097 0.02 SN38-GLY-GA 0.126 0.02 SN38 0.025 0.009Camptothecin (CPT) 0.083 0.023 CPT-SI 0.101 0.039 Irinotecan 4.426 4.654

Example 4 Human Lung Xenograft Studies on PHF-Vinca Alkaloid Conjugates

HRLN female mice with H460 tumor cells positioned subcutaneous in flankare treated with PHF-Vinca alkaloid conjugates. Tumor growth ismonitored in parallel with positive and negative controls of paclitaxel,and saline respectively. Treatment begins when tumors reach an averagesize of 80-120 mg and tumor volumes are measured twice per week untilanimals reach an endpoint tumor size of 2 grams or 45 days, whichevercomes first. Conjugates are administered as solutions in salineintravenously at dose levels of 5-50 mg/kg (expressed in Drugequivalents) on various schedules. Treatment outcomes are assessed interms of percent tumor growth delay (% TGD), defined as the percentincrease in median time to endpoint for mice treated with an agentcompared to those treated with saline, or mean or median tumor volume,for mice treated with an agent compared to those treated with saline.

Example 5 Human Colon Xenograft Studies on PHF-Non-Natural Camptothecins

HRLN female mice with HCT116 tumor cells positioned subcutaneous inflank are treated with PHF-non-natural camptothecin conjugates. Tumorgrowth is monitored in parallel with positive and negative controls ofirinotecan, and saline respectively. Treatment begins when tumors reachan average size of 80-120 mg and tumor volumes are measured twice perweek until animals reach an endpoint tumor size of 1.5 grams or 100days, whichever comes first. Conjugates are administered as solutions insaline intravenously at dose levels of 10-25 mg/kg (expressed in Drugequivalents) on a schedule of biwk×5. Treatment outcomes are assessed interms of percent tumor growth delay (% TGD), defined as the percentincrease in median time to endpoint for mice treated with an agentcompared to those treated with saline, or mean or median tumor volume,for mice treated with an agent compared to those treated with saline.The results are shown in FIG. 13.

While particular embodiments described herein have been illustrated anddescribed, it would be obvious to those skilled in the art that variousother changes and modifications can be made without departing from thespirit and scope of the disclosure. It is therefore intended to cover inthe appended claims all such changes and modifications that are withinthe scope of this disclosure.

We claim:
 1. A conjugate of Formula (I):

wherein Polyal is a polyacetal or polyketal; Linker is a dicarboxylicacid moiety containing two or more atoms between the carbonyls and

represents the two or more atoms between the carbonyl groups; R₁ is H orCH₃; R₂ is —CH(Y)—C(O)—, wherein Y is a non-hydrogen side chain of anaturally occurring amino acid; or Tether is alanine, β-alanine,sarcosine, or proline; and Drug is any organic compound with a molecularweight of between about 200 daltons and 1000 daltons, capable ofcovalent attachment to Tether; wherein when Linker is a dicarboxylicacid with two atoms between the carbonyls and Tether contains a nitrogenwith no reactive hydrogen, the release half-life of Drug is from about10 h to more than about 300 h; when Linker is a dicarboxylic acid withat least three atoms between the carbonyls and contains a heteroatomalpha to the carbonyl forming the ester, the release half-life is lessthan about 10 hours; when Linker is a dicarboxylic acid with at leastthree atoms between the carbonyls with no heteroatom alpha to thecarbonyl forming the ester, the release half-life is more than about 100hours; when Linker is a dicarboxylic acid with two atoms between thecarbonyls and Tether contains a nitrogen with a reactive hydrogen therelease half-life of Drug is from about 0.1 hours to about 24 hours; andwherein the release half-life being measured in 0.05M phosphate buffer,0.9% saline, pH 7.4, at 37° C.
 2. A conjugate of the Formula (II):

wherein X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of the CH₂is optionally substituted; R₁ is H or CH₃; R₂ is —CH(Y)—C(O)—, wherein Yis a non-hydrogen side chain of a naturally occurring amino acid; orTether is alanine, β-alanine, sarcosine, or proline; Polyal is apolyacetal or polyketal; Drug is any organic compound with a molecularweight of between about 200 daltons and 1000 daltons, capable ofcovalent attachment to Tether; wherein when Linker is a dicarboxylicacid with two atoms between the carbonyls and Tether contains a nitrogenwith no reactive hydrogen, the release half-life of Drug is from about10 h to more than about 300 h; when Linker is a dicarboxylic acid withat least three atoms between the carbonyls and contains a heteroatomalpha to the carbonyl forming the ester, the release half-life is lessthan about 10 hours; when Linker is a dicarboxylic acid with at leastthree atoms between the carbonyls with no heteroatom alpha to thecarbonyl forming the ester, the release half-life is more than about 100hours; when Linker is a dicarboxylic acid with two atoms between thecarbonyls and Tether contains a nitrogen with a reactive hydrogen, therelease half-life of Drug is from about 0.1 hours to about 24 hours; andwherein the release half-life being measured in 0.05M phosphate buffer,0.9% saline, pH 7.4, at 37° C.
 3. The conjugate of claim 2, whereinPolyal is a polyacetal.
 4. The conjugate of claim 2, wherein Polyal is apolyketal.
 5. The conjugate of claim 3, wherein the polyacetal ispoly(hydroxymethylethylene hydroxymethylformal) (PHF).
 6. The conjugateof claim 2, wherein R₁ is H.
 7. The conjugate of claim 2, wherein R₁ isCH₃.
 8. The conjugate of claim 2, wherein R₂ is —CH(Y)—C(O)—, and Y is anon-hydrogen side chain of a naturally occurring amino acid.
 9. Theconjugate of claim 2, wherein X is —CH₂—.
 10. The conjugate of claim 2,wherein X is —OCH₂—.
 11. The conjugate of claim 2, wherein X is—CH₂CH₂—.
 12. The conjugate of claim 2, wherein X is optionallysubstituted with a C₁-C₆ alkyl group.
 13. The conjugate of claim 2,wherein Tether is an amino acid selected from alanine, β-alanine,sarcosine, and proline.
 14. The conjugate of claim 2, wherein Drug isfumagillol.
 15. The conjugate of claim 2, wherein Drug is a vincaalkaloid.
 16. The conjugate of claim 2, wherein Drug is a non-naturalcamptothecin.
 17. The conjugate of claim 16, wherein the non-naturalcamptothecin is SN38.
 18. The conjugate of claim 17, wherein theconjugate is selected from the group consisting of

wherein k ranges from 1 to 30, m ranges from 0 to 300, and n ranges from100 to 750, and wherein the polyal comprises randomly distributedcovalently bound monomer blocks shown in brackets; and pharmaceuticallyacceptable salts thereof.
 19. A conjugate of the Formula (III):

or a pharmaceutically acceptable salt thereof; wherein Polyal is apolyacetal or polyketal; X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one ormore of —CH₂— is optionally substituted; R₁ is H or CH₃; R₂ is—CH(Y)—C(O)—, wherein Y is a non-hydrogen side chain of a naturallyoccurring amino acid or —NR₁R₂— is alanine, β-alanine, sarcosine, orproline; R₇ is —CH₃ or —CHO; and R₈ is —OCOCH₃ or OH.
 20. A conjugate ofthe Formula (IV):

or a pharmaceutically acceptable salt; wherein Polyal is a polyacetal orpolyketal; X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂—is optionally substituted; R₁ is H or CH₃; R₂ is —CH(Y)—C(O)—, wherein Yis a non-hydrogen side chain of a naturally occurring amino acid; or—NR₁R₂— is alanine, β-alanine, sarcosine, or proline; R₃ is —H, —Cl, —F,—OH or alkyl; or R₃ and R₄, may be taken together to form a five- orsix-membered ring; R₄ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl,—CH₂CH₂Si(CH₃)₃, or —Si((CH₃)₂)-t-Butyl; R₅ is —CH₂—N(CH₃)₂, NH₂, orNO₂; R₆ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or—N-4-methylcyclohexylamine; or R₅ and R₆, may be taken together to forma six-membered optionally substituted ring; p is 0 or 1; and q is 0or
 1. 21. A pharmaceutical composition comprising a conjugate of theFormula (III):

or a pharmaceutically acceptable salt thereof; wherein Polyal is apolyacetal or polyketal; X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one ormore of —CH₂— is optionally substituted; R₁ is H or CH₃; R₂ is—CH(Y)—C(O)—, wherein Y is a non-hydrogen side chain of a naturallyoccurring amino acid; or —NR₁R₂— is alanine, β-alanine, sarcosine, orproline; R₇ is —CH₃ or —CHO; and R₈ is —OCOCH₃ or OH; and apharmaceutically acceptable carrier.
 22. A method of treating cancer,comprising administering to a subject in need thereof a polyal-vincaalkaloid conjugate of Formula (III) of claim 19 or a pharmaceuticallyacceptable salt thereof, in an amount effective to treat the cancer. 23.The method of claim 22, wherein the polyal-vinca alkaloid conjugate ofFormula III is a PHF-vinca alkaloid conjugate.
 24. The method of claim22, wherein the cancer is selected from the group consisting of anal,astrocytoma, leukemia, lymphoma, head and neck, liver, testicular,cervical, sarcoma, hemangioma, esophageal, eye, laryngeal, mouth,mesothelioma, skin, myeloma, oral, rectal, throat, bladder, breast,uterus, ovary, prostate, lung, colon, pancreas, renal, and gastric. 25.A pharmaceutical composition comprising a conjugate of Formula (IV):

or a pharmaceutically acceptable salt; wherein Polyal is a polyacetal orpolyketal; X is —CH₂—, —OCH₂—, or —CH₂CH₂—, wherein one or more of —CH₂—is optionally substituted; R₁ is H or CH₃; R₂ is —CH(Y)—C(O)—, wherein Yis a non-hydrogen side chain of a naturally occurring amino acid; or—NR₁R₂— is alanine, β-alanine, sarcosine, or proline; R₃ is —H, —Cl, —F,—OH or alkyl; or R₃ and R₄, may be taken together to form a five- orsix-membered ring; R₄ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl,—CH₂CH₂Si(CH₃)₃, or —Si((CH₃)₂)-t-Butyl; R₅ is —CH₂—N(CH₃)₂, NH₂, orNO₂; R₆ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or—N-4-methylcyclohexylamine; or R₅ and R₆, may be taken together to forma six-membered optionally substituted ring; p is 0 or 1; and q is 0 or1; and a pharmaceutically acceptable carrier.
 26. A method of treatingcancer, comprising administering to a subject in need thereof apolyal-non-natural camptothecin conjugate of the Formula (IV) of claim20 or a pharmaceutically acceptable salt thereof, in an amount effectiveto treat the cancer.
 27. The method of claim 26, wherein thepolyal-non-natural camptothecin of Formula IV is a PHF-non-naturalcamptothecin conjugate.
 28. The method of claim 27, wherein thePHF-non-natural camptothecin conjugate is PHF-SN38.
 29. The method ofclaim 26, wherein the cancer is selected from the group consisting ofanal, astrocytoma, leukemia, lymphoma, head and neck, liver, testicular,cervical, sarcoma, hemangioma, esophageal, eye, laryngeal, mouth,mesothelioma, skin, myeloma, oral, rectal, throat, bladder, breast,uterus, ovary, prostate, lung, colon, pancreas, renal, and gastric. 30.The conjugate of claim 19, wherein the conjugate is selected from thegroup consisting of